10:15 A – 8:30 P
Cell culture
- Fix Kc cells
- cells have lost adherent properties all washed off. Cells still not attached to new passaged flask either. Probably time to restart this stock from frozen anyway.
DNA in situs
- En 10 uL probe, 1.5 uL secondary 1.5 uL tertiary in 50 uL hybe mix. Split between embryos and cells
- imaged En expressing cells of embryos prior to staining (coverslip 3, previous 2 insufficent embryos).
- can see DAPI signal through pentaband at 100x in focus with 5% + laser
- G ~15 uL probe (all), 1.5 uL secondary 1.5 uL tertiary in 20 uL hybe mix.
- using 5/28 prepared fixed & permiabilized cells.
- front right block downstairs set at 84, thermo in water under glass measuring ~78C. Denatured 2.5 min.
- incubating samples at 45C O/N.
chromatin
- analysis of remaining K from 6-10 data
- finish O/N STORM of Y spots with feducials
- set up new O/N STORM of B spots with feducials (using 200 nm beads MUCH brighter, require very little 561) at 1:20,000 (probably should be higher, not that many stuck).
- explore alternate image based drift correction for B data: imaged based autocorrelation still works best, just need to be able to tune binning and timestep parameters and see plots. Also nice to be able to run free-standing easily.
Coding
- Wrote fast fully-matlab implementation of correlation based drift correction.
- running batch correction of serious drift in B data. Prime key is just a longer integration interval, but it helps a lot to be able to see how much structure is in the auto-correlation function and what the actual drift traces look like.
- zooming in on the centroid of the autocorrelation function speeds up algorithm and reduces potential for errors due to off peak maxima.